Mini-Review

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Non–HDL cholesterol is simply defined as the difference between total and HDL cholesterol and constitutes all the potentially proatherogenic apolipoprotein B (apoB)-containing particles [primarily VLDL, intermediate-density lipoprotein (IDL), and LDL as well as chylomicron remnants and lipoprotein(a)].³ Its measurement has an advantage over direct LDL measurement as a more comprehensive ASCVD risk marker as the latter excludes the cholesterol of TGRLs, which are proatherogenic.

Thus, for patients with diabetes having mixed dyslipidemia, calculated LDL cholesterol might not be an adequate index of overall lipid-associated risk with non-HDL providing additional information over LDL cholesterol alone. Owing to its simple calculation, the non–HDL cholesterol level is easily available to the physician with every lipid profile report without any additional cost. Since it excludes the measurement of TG, it avoids the possible downside of TG as a marker of CHD risk and directly reflects the cholesterol content of all particles that may be proatherogenic. Furthermore, its derivation does not need an LP to be done in the fasting state, and it avoids the possible imprecision related with the intra-individual variability of triglyceride measurements. In the Strong Heart Study, a long-term follow-up of patients with diabetic dyslipidemia, non- HDL cholesterol was a stronger predictor of cardiovascular disease than other lipid parameters, e.g., total/HDL cholesterol used earlier.^3,4

Notwithstanding the subtle differences in different clinical practice guidelines, the fundamental objectives remain the same for LP testing, i.e., to prevent ASCVD in healthy subjects (primary prevention) and to provide early detection and retard the progression of disease in those with established ASCVD (secondary prevention). It becomes necessary in a patient with a family history of premature death due to myocardial infarction in first degree relatives (<55 years in male and <65 years in female), history of familial hypercholesterolemia or patient with clinical stigmata of familial hypercholesterolemia (xanthoma/ xanthomata or eyelid xanthelasma), patients with comorbidities that are likely to be benefitted from statin use, i.e., patients with diabetes, chronic kidney disease, HIV, etc. and risk stratification for patients without the disease, who might benefit from hypolipidemic drug treatment.⁵

1 mL of venous blood is collected from the antecubital vein and in a gel-barrier transport, green-top (heparin) or lavender-top ethylenediaminetetraacetic acid (EDTA) vial. Sample should be preserved at ambient room temperature and serum (gel barrier tubes) or plasma (heparin/EDTA vial) obtained after centrifugation is allowed to separate within 45 minutes. The universal recommendation is that patient should remain fasting for more than 8 hours (preferably 12 hours of complete dietary restriction with exception to water and medicines) but a non-fasting sample would suffice for total cholesterol and HDL-c estimation, diagnosis of metabolic syndrome and CV risk estimation in patients being considered for primary prevention. The reason being the utility of LDL-c estimation is limited apart from the management of familial hyperlipidemia. Most of the risk estimators meant for cardiovascular diseases, e.g., Reynolds risk score, Framingham risk score, ASCVD pooled cohort risk estimator, etc., take into account only total cholesterol and HDL-c among all risk factors and these components have negligible variation whether the patient is fasting or in fed-state. Fasting sample becomes mandatory in cases of suspected genetic causes of hyperlipoproteinemia, premature ASCVD, hypertriglyceridemia and in those at risk of pancreatitis. The rationale behind fasting is to avoid a post-meal surge of triglycerides, and reference values that we measure were worked out on fasting blood samples. Moreover, non-HDL cholesterol (total cholesterol − HDL cholesterol), a secondary target of lipid-lowering therapy can also be estimated in the non-fasting sample.⁶

In addition to food intake, certain other factors also affect the LP estimation that needs consideration prior to interpretation of the test results. Applying a tourniquet too long (2–5 mins) prior to sample collection can falsely elevate cholesterol levels by 5-15%. It is observed that there is the effect of seasonal variation on lipid profile, e.g., in winter the cholesterol is found to be little increased and the same happens for triglycerides in summer. When one assumes supine from an upright posture there is a pooling of blood and the resultant dilutional effect cab bring down triglycerides by 15% and cholesterol by 10%. Nephrotic syndrome can cause hypercholesterolemia including total, LDL and VLDL fractions and the same happens in hypothyroidism also. In any acute infection or inflammatory states, there is an alteration of the usual lipid profile with the triglycerides going high and cholesterol coming down. The changes usually become apparent within the first 24-48 hours after MI, become most obvious in 4-7 days, and then come to the baseline after some months when we can re-estimate the lipid profile.⁷

Total cholesterol is measured by the cholesterol oxidase phenol 4-amino antipyridine method (CHOD-PAP) where cholesterol esters are hydrolyzed and then oxidized to produce a red hue. After incubation of the resultant at 37° C for 10 minutes, absorbance from the quinoneimine dye is measured at 505 nm. Other estimation methods are the Lieberman Burchat method and the Sulkowski method. Triglycerides are measured with glycerol-3-phosphate oxidase-phenol-4-amino pyrophosphate (GPO-PAP) method. It is hydrolyzed and oxidized to produce a red colour; the absorbance of which is measured similarly as before. LDL, VLDL and chylomicrons are precipitated by the addition of polyethylene glycol (PEG) 6000. After centrifugation HDL remains in the supernatant and is measured with the CHOD-PAP method as described earlier. The LDL cholesterol estimation can be done in two ways; indirect and direct. The indirect approach is based on the Friedewald cholesterol estimation formula.⁵ The formula is as follows